The invention relates to the use of dielectrophoresis to levitate, in three-dimensions, a neutral particle such as a biological cell. More particularly, the invention relates to such a use of dielectrophoreses wherein a unique combination of a particular electrode configuration and an active feedback control is utilized to obtain more precise dielectric properties of the particle.
The need exists for methods of characterizing particles, particularly biological cells or their parts (e.g., organelles, ghosts, etc.). Such individual particles have unique characteristics and their identification and observation can be a powerful analysis tool to facilitate the study of the particles. In this regard, there is a particular need for a means to microscopically observe the characteristics of individual cells. That is, although different cells have different characteristics, when a number of cells are observed and analyzed simultaneously, there tends to be a masking of certain characteristics of the individual cells. The microscopic observation and analysis of a single cell would be particularly useful in the area of cancer diagnosis.
Non-uniform fields and in particular, dielectrophoretic methods have previously been used to separate and analyze biological cells. See e.g., U.S. Pat. Nos. 4,326,934 and 4,661,451, the contents of which are hereby incorporated by reference. Dielectrophoresis has been defined as the motion of a neutral particle due to the action of a non-uniform electric field on its permanent or induced dipole moment. When a particle is introduced into a system with a nonuniform electric field, the field induces a dipole in that particle. The divergent non-uniform nature of the field results in one end of the dipole being in a region of higher field strength than the other. The effect is that the dipole is pushed in the direction of the increasing field.
Non-uniform electric fields can induce translational and rotational motions of cells in suspension. The nonuniform field acts by aligning or inducing a dipole moment in the cell. The cell is then impelled by the field non-uniformity, usually towards the region of greatest field intensity.
The force created is known as the dielectrophoretic force, and the resulting motion dielectrophoresis. In the event the cell being acted upon is suspended in a polarizable medium, the net polarization of the whole may be such as to evoke a dielectrophoretic force in favor of pushing the body either into or away from the region of higher field intensity. The cell experiences "positive" dielectrophoresis when it is forced into the region of higher field intensity; "negative" dielectrophoresis results when the cell is pushed away from the region of higher field intensity.
It is well-known that a neutral particle, when subjected to the influence of a nonuniform, time varying (AC) electric field, may exhibit one of the following behaviors:
(1) Positive dielectrophoresis, i.e., attraction toward the region of high field intensity; PA1 (2) Negative dielectrophoresis, i.e., repulsion toward the region of lower field intensity; or PA1 (3) Zeresis, i.e., no net displacement. PA1 (i) providing a cell suspension in a levitation chamber of a dielectrophoresis apparatus containing an electrode system, the suspension being provided between the electrodes of the system; PA1 (ii) subjecting a cell from the suspension to a nonuniform electric field generated from voltage applied to the electrodes of the electrode system, wherein there is established a non-uniform gradient that is positive along the axis extending between the electrodes and that is negative along the radial direction, thereby reducing radial migration of the cell; PA1 (iii) dynamically levitating said cell in three-dimensions; PA1 (iv) monitoring the position of the cell; PA1 (v) providing a focussed cell by maintaining or adjusting the position of the cell by controlling the voltage applied to the electrode system, wherein steps (iv) and (v) are carried out using an active feedback control means.
These processes arise from the following sequence of phenomena. The electric field induces a charge separation or dipole in the neutral particle. The resultant dipole consisting of equal numbers of slightly separated positive and negative charges now experiences a net force upon it because of the non-uniformity of the electric field. One or the other of the charge sets will be in a weaker electric field. Since the force upon a charge is exactly dependent upon the amount of charge, and upon the local field acting upon that charge, it will be seen that a net force arises upon the particle, despite the fact that it is neutral overall and has no excess charge of either type. The same considerations apply to the supporting fluid medium. The net of these dielectrophoretic forces upon the particle and its supporting medium acts to impel the particle toward the stronger field in positive dielectrophoresis. If, on the other hand, the net force upon the particle and medium is such as to impel the particle toward the region of weaker field, negative dielectrophoresis results.
In electrophoresis, the field induced motion of charged particles, the direction of the force is dependent upon the sign of the charge and upon the direction of the field. However, in dielectrophoresis, the force depends upon the square of the field intensity, and is independent of the direction of the field. For this reason, dielectrophoresis works well in AC fields. For a particle to experience either positive or negative dielectrophoresis it must be subject to a divergent electric field.
"Stable" levitation of a particle in a medium can be obtained with the use of a divergent electric field only if the time-average of the dielectrophoretic force at any point is constant, and if the thrust by the dielectrophoretic forces tend to push the particle into a region where the dielectrophoretic force is weaker. For example, if this dielectrophoretic force is to be balanced against gravity, then the field must weaken or diverge in the upward direction. Moreover, having the dielectrophoretic force on the particle lie in the direction away from the region of higher field intensity requires that negative dielectrophoresis be possible, i.e., that the effective time-average permittivity of the particle be less than that of the medium.
"Dynamic" levitation, on the other hand, means the suitable repetitive application of controlled positive dielectrophoresis such that essentially or nearly static localization of the particle is obtained. As an example, one could suspend a living cell in an aqueous medium below a pointed electrode by continually monitoring and adjusting the upward force to prevent the cell from falling away, yet not reaching and sticking to the upper electrode, nor touching the lower flat electrode.
Previously, there was developed a method for the dynamic dielectric levitation of living individual cells. See K. Kaler an H. Pohl, "Dynamic Dielectrophoretic Levitation of living Individual Cells", IEE Transactions on Industry Applications, Vol. 1A, 6, November/December 1983, the contents of which is hereby incorporated by reference. That method was used to characterize individual cells. In the method, lone live cells were levitated by means of a dielectrophoretic force. This was done by observing the cell through a microscope and manually adjusting the voltage applied to the electrodes of the system to stabilize the cells in the electric field. The relative polarization of the cells and their aqueous support medium was then determined. When repeated over a range of frequencies, a spectrum of dielectric (polarization) responses was obtained which was used to characterize a single living cell. Unfortunately, although this method was a great advance in the art, it suffered from a variety of serious drawbacks. In particular, the manual nature of the method was inconvenient and imprecise. For example, for each run the spectrum of dielectric responses differed, thus adversely effecting the ability of the system to obtain reproducible results since different results were obtained every time. Also, it was difficult, if not impossible, to obtain suitably focussed cells because the cells were found to migrate radially in the field generated in that system.